This application is related to Japanese Patent Applications Nos. 2001-094878 filed in Mar. 29, 2001 and 2001-182085 filed in Jun. 15, 2001, whose priorities are claimed under 35 USC xc2xa7119, the disclosures of which are incorporated by reference in their entirety.
1. Field of the Invention
The present invention relates to a flow cytometer for optically detecting and analyzing particles such as blood cells in blood, material components contained in urine or the like.
2. Description of the Related Art
Various particle analyzers have been developed for automatically detecting and analyzing particles contained in a specimen, for example, blood cells in blood such as red blood cells, white blood cells and blood platelets, or material components in urine such as bacteria, blood cells, white blood cells, epithelial cells or casts. A flow cytometer is well known as such a particle analyzer. The flow cytometer includes a detecting section with a flow cell, a laser and a photoelectric conversion element. The flow cell surrounds a sample liquid containing particles to be analyzed with a sheath fluid to converge the sample liquid into a thin flow so that the particles align and pass therethrough. The laser irradiates the passing particles with a laser beam to obtain light generated from each particle i.e., optical information. The optical information includes scattered light such as forward scattered light, sideward scattered light, backward scattered light, fluorescence or the like. The optical information is suitably selected depending upon an analyzed target. The photoelectric conversion element detects the optical information to generate a pulsed electrical signal.
A waveform of the electrical signal obtained as mentioned above is processed to calculate a parameter representing the characteristics of each particle and the particles to be analyzed are classified and counted based on the parameter.
In the optical information, an intensity of the light is recognized as a height (peak level) of the signal waveform, and at the same time, a light emitting period (pulse width) is clocked. That is, the above parameter includes the peak level and the pulse width. For example, the peak level of the signal of the forward scattered light (hereinafter referred to as a xe2x80x9cparticle signalxe2x80x9d) represents a size of a particle, while the pulse width represents a length of a particle. In case where a fluorescent staining is performed in advance to particles, for example, nucleated cells, a fluorescent signal can be obtained from each particle. The peak level of the signal represents a staining degree of the nuclear or the like, while the pulse width represents a length of the fluorescent staining portion. A histogram, formed based upon the parameter representing the characteristics of each particle or a scattergram showing a distribution of the particles, is formed by combining a plurality of parameters, whereby the type or number of the particles contained in the sample is statistically analyzed.
In recent years, the above-mentioned flow cytometer generally utilizes a laser diode from which the laser beam is irradiated to the flow cell.
FIG. 1 is a schematic view showing one example of an optical system in a conventional flow cytometer using a laser diode 1 for a light source. A sample liquid T containing particles to be analyzed is supplied from a nozzle 11 into the flow cell 6 in the direction shown by an arrow A. A sheath liquid S is supplied to the flow cell 6 for surrounding the supplied sample liquid T, whereby the sample liquid T is converged into a thin sample flow by a hydrodynamic effect. As a result, the sample flow is passed through the flow cell 6 with the particles aligned. A radiant laser beam LB emitted from the laser diode 1 is collimated by a collimator lens 3, and then, passes through a cylindrical lens 4 and a condenser lens 5 to form a beam spot at a position R of the flow of the sample liquid T in the flow cell 6.
As shown in FIG. 2, the laser diode 1 has inherent features that the laser beam LB is diffusible and has an elliptic cross section. Thus, the laser beam LB emitted from the laser diode 1 along an optical axis in the direction of Z has radiation angles defined by a large angle xcex81 in the direction of a major diameter of the ellipse, i.e., in the direction of X, and a small angle xcex82 in the direction of a minor diameter of the ellipse, i.e., in the direction of Y.
In FIG. 1, increasing the radiation angle xcex8 of the laser beam with respect to the flowing direction of the sample liquid T, that is, increasing a collimated beam width d in the same direction brings the following merits:
1. The amount of light of the beam spot at the position R can be increased.
2. The diameter of the beam spot in the flowing direction of the sample liquid T can be decreased in view of a diffraction limit of the beam, since the laser beam is such a Gaussian beam that the Gaussian beam of a larger diameter is less divergent than that of a smaller diameter.
These merits bring the effects of enhancing detecting sensitivity and preventing simultaneous illumination to a plurality of particles. Therefore, in the conventional flow cytometer, the laser diode 1 is mounted so that the major diameter of the elliptic section of the laser beam LB is arranged so as to be parallel to the flowing direction of the sample liquid T in the flow cell 6.
However, the above-mentioned arrangement causes a significant demerit. FIG. 3 shows a relationship between such as an arrangement and an intensity of the detected forward scattered light signal. In FIG. 3, the laser beam LB has such a large radiation angle xcex8 in the direction parallel to the flowing direction of the sample liquid T, that the laser beam LB is partially kicked at upper and lower edges 3a and 3b of the collimator lens 3 to generate stray beams SB1 and SB2. Therefore, the stray beams SB1 and SB2 are focused on focal points BS1 and BS2 above and below a beam spot BS0 focused by a main beam MB in the flow of the sample liquid T. As a result, signals S1 and S2 (hereinafter referred to as xe2x80x9cstray beam signalxe2x80x9d) attributed to the stray beams SB1 and SB2 are detected in addition to a particle signal S0 due to the main beam MB.
The stray signals S1 and S2 are mistakenly detected as small particle signals, which have a bad influence on the counting and classifying result of the particles to be analyzed. This demerit also applies to each detection of a sideward scattered light signal, backward scattered light signal and fluorescent signal, besides the detection of the forward scattered light signal.
To overcome the above-mentioned problems, the following attempt in a signal processing system is taken. In the system, a threshold value Vth is set to a value so as to detect the particle signal S0 but the stray beam signals S1 and S2 as shown in FIG. 4. That is, the xe2x80x9cthreshold value Vthxe2x80x9d here is used for selecting the particle signal S0 in a series of signals S1, S0, S2. The signal intensity (peak level) VP and the light emitting period (pulse width) PW are calculated based on a part of the signal which exceeds the threshold value Vth.
In case where there is a great difference in pulse size between the particle signal S0 and the stray beam signals S1 and S2 (each of the stray beam signals S1 and S2 has a pulse smaller than that of the particle signal S0). For example, in case a relatively large-sized particle such as a blood cell is a particle to be detected, higher setting of this threshold value Vth enables to detect only the particle signal S0 and not to detect the stray beam signals S1 and S2. Accordingly, the laser diode 1 is provided in the conventional general flow cytometer such that the major diameter of the elliptic section of the emitted laser beam LB is arranged so as to be parallel to the flowing direction of the sample liquid T in the flow cell 6, whereby the above-mentioned merits (1. increasing the amount of light at the beam spot; and 2. decreasing the diameter of the beam spot) can be enjoyed.
However, when the sample includes particles of various size (for example, a diameter of about 0.5 to 100 microns), the threshold value Vth is required to be set lower corresponding to the minimum particle signal S0. Therefore, the stray light signals S1 and S2 exceeds the threshold value Vth and are detected by mistake as small-sized particles. This causes a miscount of the particles or gives an adverse affect to the pulse width information of the particle signals. For example, urine contains bacteria, red blood cells, white blood cells, epithelial cells, casts or the like, each of which has a various size. Particularly, the bacteria is greatly smaller than the other particles in most cases. In case of detecting these particles, the threshold value Vth for detecting the particle signal S0 should be set lower in order to detect the bacteria. Therefore, the stray beam signals S1 and S2 generated with the particle signal S0 of the white blood cell brings an analysis result as if the bacteria is also detected, although only the white blood cell should originally be detected. Further, the pulse width PW of the particle signal S0 of the white blood cell becomes greater than the original width, whereby the white blood cell may be analyzed as another type.
As described above, the adverse affect of the stray beam signals S1 and S2 is more remarkable in a flow cytometer that is required to detect not only large-sized particles but also small-sized particles.
If an attempt in the optical system is made for overcoming the above-mentioned problem, a laser diode having a narrower radiation angle is considered to be selected as the laser diode 1 in the optical system shown in FIG. 3 so that the laser beam LB does not impinge on the upper and lower edges 3a and 3b of the collimator lens 3. However, the laser diode generally has a feature that the radiation angle is determined by the wavelength of the laser beam. Further, the wavelength of the laser beam is necessarily determined by the character or kind of a particle to be analyzed. Accordingly, the laser diode having a narrow radiation angle of the laser beam cannot freely be selected.
For example, supposing that a fluorescent staining is performed in advance to blood cells in blood or material components in urine, obtaining fluorescent information from a fluorescent portion of each particle requires a laser beam having a relatively short wavelength, e.g., 700 nm or less. However, the laser diode generally has a characteristic that, the shorter the wavelength of the emitted laser beam becomes, the wider the radiation angle becomes. When a laser diode that emits a laser beam of relatively short wavelength is obliged to be used, the radiation angle of the laser beam inevitably increases. Therefore, the laser beam is partially kicked at the upper and lower edges 3a and 3b of the collimator lens 3, thereby causing the problem of generating the stray beam signals SB1 and SB2.
Bringing the laser diode 1 close to the collimator lens 3 or using a collimator lens having a large diameter is considered as another attempt for preventing the laser beam from impinging on the upper and lower edges 3a and 3b of the collimator lens 3.
In such a case, the collimator lens 3 is required to have a greater NA in order to bring the laser diode 1 close to the collimator lens 3. However, when a spherical lens is used as the collimator lens 3, there is a physical limit upon increasing NA with the lens diameter kept constant.
To use a collimator lens having a greater diameter, the lens is required to increase its diameter with the same curvature (i.e., a collimator lens having a greater NA is required to be used). However, there is also a physical limit upon increasing a lens diameter without changing the curvature.
On the other hand, it is not preferable to use an aspherical lens as the collimator lens 3. This is because the aspherical lens is unsuited to the collimator lens 3 from the viewpoint of the level of the current lens manufacturing technique.
There is another attempt in which a slit is provided between the laser diode 1 and the collimator lens 3 for limiting the radiation angle of the laser beam so as not to prevent the laser beam from impinging on the upper and lower edges 3a and 3b of the collimator lens 3. However, this attempt causes a great loss of the amount of beam. Further, the laser beam contacting edges of the slit is scattered, so that stray beams are likely to occur.
Accordingly, the above-mentioned problems cannot be perfectly solved by the aforementioned attempts.
The present invention is accomplished in view of the above subjects, and aims to provide a flow cytometer that prevents the generation of the stray beam signals by devising the angle at which the laser diode is arranged, whereby only the particle signal due to the main beam can effectively be detected.
The present invention provides a flow cytometer including a flow cell for flowing a sample liquid in a flowing direction to form a sample flow, the sample liquid containing particles to be analyzed, a laser diode radiating a laser beam having an elliptic cross section, a beam collimating section for collimating the laser beam radiated from the laser diode, a beam spot forming section for focusing the collimated beam at the sample flow in the flow cell to form a beam spot, and a light receiving section for receiving light generated from the particles at the beam spot to detect optical information of the particles, wherein the laser diode is arranged such that a minor diameter of the elliptic section of the laser beam is parallel to the sample flow.